The development of CDNs including ZFNs and TALENs has made it possible to perform precise genetic engineering in many cell types and species (Bibikova et al., 2003; Christian et al., 2010; Hockemeyer et al., 2009; Kim et al., 1996; Meyer et al., 2010; Moehle et al., 2007; Porteus and Baltimore, 2003; Urnov et al., 2010). These are hybrid endonucleases consist of a Fokl nuclease domain and a DNA binding domain assembled from optimized DNA binding modules that are specific for either single nucleotide (for TALENs) or trinucleotide motifs (for ZFNs). Once introduced in cells, these enzymes generate a DSB in the genome at or near the desired modification site. This event induces the natural DDR process to mend the breakage (Rouet et al., 1994). The primary pathway is NHEJ in which the two ends are processed and ligated together with nucleotide insertions and deletions. Though highly efficient, this mechanism can only produce functional knockout alleles that are often heterogeneous and difficult to isolate without clonal selection. Accurate gene modification relies on HR in which exogenous DNA fragments flanked by homology sequences around the DSB site can be copied faithfully from a template with defined boundaries (Rouet et al., 1994).
Targeted gene modification has been almost exclusively mediated by homologous recombination. Though CDNs have made gene targeting feasible in cell lines that have low intrinsic HR efficiency, NHEJ is still the dominant DSB repair pathway. As a result, KO alleles generated by NHEJ are obtained at a much higher frequency than KI alleles generated by HR. A deficiency in NHEJ can significantly promote HR as seen in E. coli in which NHEJ is neglectable (Liang et al., 1996; Maresca et al., 2010).